Rice receptor-like kinase oslike1 and its application in resistance to magnaporthe oryzae

Abstract

The application discloses a rice receptor-like kinase OsLIKE1 and application thereof in Magnaporthe oryzae resistance. The application finds for the first time that the receptor-like kinase OsLIKE1 regulates rice resistance to Magnaporthe oryzae through phosphorylation. Transgenic plants with overexpressed OsLIKE1 gene in rice have significant resistance to Magnaporthe oryzae, and thus can be used for cultivation of Magnaporthe oryzae-resistant rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a rice receptor kinase OsLIKE1 and its application in rice blast fungus resistance. Background Technology

[0002] Rice, as the most widely cultivated and most important food crop globally, is the staple food for more than half of the world's population. With the rapid increase in the global population, the demand for and security of rice production have become increasingly important. Among the many diseases affecting rice, rice blast, caused by Magnaphaltheoryzae, is the most prevalent and devastating fungal disease in rice-producing areas worldwide, seriously threatening global food security. Currently, the control of this disease mainly relies on breeding resistant varieties and chemical control. Chemical control has many drawbacks, such as high costs, the continuous development of fungal resistance leading to poor efficacy, and environmental pollution. Regarding the breeding of resistant varieties, due to the genetic diversity and virulence variability of Magnaphaltheoryzae during evolution, resistant varieties lose their resistance after 3-5 years of promotion, rendering them unusable. Therefore, a deeper understanding of the molecular mechanisms of the interaction between rice and rice blast fungus is not only helpful in developing and applying broad-spectrum and effective control strategies against rice blast fungus, but also provides a theoretical basis for exploring new gene resources for breeding rice blast fungus, which has important application value for the sustainable production of rice in the future.

[0003] In the long-term interaction, selection and co-evolution between plants and pathogens, a complex and precise arms race of attack, defense, re-attack and re-defense has been taking place between the two. In order to resist the infection of pathogens, plants have evolved two levels of immune systems[1]. The first level of immune response is the immune response triggered by the recognition of pathogen-associated molecular pattern (PAMP) by pattern recognition receptors (PRRs) located on the cell membrane (PAMP-triggered immunity, PTI). However, in the long-term co-evolution of plants and pathogens, pathogens have evolved a strategy to suppress PTI in order to escape the recognition of the host. Pathogens secrete effectors into plant cells, affecting the physiological and biochemical metabolism of plants, inhibiting the plant's defense response and promoting their own infection. However, plants have evolved intracellular receptors to recognize pathogen effectors and stimulate immune response (effector-triggered immunity, ETI), which is the second level of immune response in plants[2]. PTIs triggered by PAMPs are stable, persistent, and broad-spectrum. Explaining their mechanism has important theoretical and practical value for improving the long-lasting disease resistance of crops.

[0004] Receptor-like kinases (RLKs) are a type of pattern recognition receptors (PRRs) on the plant surface. They consist of an intracellular serine / threonine-containing protein kinase domain, a transmembrane domain, and an extracellular binding domain [3]. RLKs play a key role in plant PTIs. Currently, the RLKs that have been studied relatively well include FLS2, EFR, XA21, and CORE. Among them, the most studied PRR is the leucine-rich receptor-like kinase FLS2 in Arabidopsis thaliana. It can recognize flg22 (a 22-residue polypeptide of bacterial flagellin that is conserved in evolution), activate the co-receptor BAK1, and transmit disease resistance signals downward through BAK1 / BIK1, thereby triggering a series of disease resistance responses [4]. EFR, as one of the members of the LRR-RK family group XII, belongs to the same group as FLS2, and its structure is very similar to that of FLS2 [5]. EFR can specifically recognize the 18-amino acid short peptide (elf18) at the N-terminus of elongation factor (EF-Tu), thereby stimulating downstream immune responses. Studies have shown that EFR's specific recognition of elongation factor (EF-Tu) is only found in cruciferous plants [6]. In rice, LRR receptor kinase XA21 forms a co-receptor with Arabidopsis thaliana BAK1 homolog SERK2, and rice XA21 triggers an immune response by recognizing the 17-amino acid sequence at the N-terminus of Ax21 in Bacteroides oryzae [7]. Recent studies have shown that LRR-RK CORE in tomato is essential for recognizing csp22, and csp22 directly binds to CORE to exert its function [8]. Another study also showed that the homolog of CORE in Nicotiana benthamiana, NbCSPR, is also essential for recognizing csp22 [9]. Similar to BAK1, chitin-induced receptor kinase 1 (CERK1), as a member of the RLK family of proteins, is a regulatory receptor kinase that binds to different LysM-containing PRRs to activate immune signals. In rice, the LysM-RLP chitin-binding protein (CEBiP) forms a homodimer after binding to chitin, and then forms a heterodimer with CERK1 to perform RLK function [10-11]. The other two LysM-containing RLPs are LYP4 and LYP6, which act as bispecific receptors for chitin and peptidoglycan, forming co-receptors with rice CERK1 in a ligand-dependent manner to play a role in signal transduction [12-13].

[0005] Currently, bioinformatics analysis has predicted the existence of over 1100 receptor-like kinases (RLKs) in rice. Although the functions and mechanisms of action of some RLKs have been reported, the mechanisms of action of many resistance-related RLK molecules in rice remain unclear. Therefore, identifying and cloning resistance-related RLKs is of significant guiding value for a deeper understanding of rice disease resistance mechanisms and the discovery of rice disease resistance gene resources.

[0006] References

[0007] 1. Ramirez-Prado JS, Abulfaraj AA, Rayapuram N, et al. Plant immunity: from signaling to epigenetic control of defense. Trends in Plant Science. 2018, 23: 833 - 844.

[0008] 2. Dodds PN, Rathjen JP. Plant immunity: towards an integrated view of plant - pathogen interactions. Nature Reviews Genetics. 2010, 11: 539 - 548.

[0009] 3. Shiu SH, Bleecker AB. Receptor - like kinases from Arabidopsis from a monophyletic gene family related to animal receptor kinases. Proceedings of the National Academy of Sciences, 2001, 98: 10763 - 10768.

[0010] 4. Bigeard J, Colcombet J and Hirt H. Signaling mechanisms in pattern - triggered immunity (PTI). Molecular Plant. 2015, 8: 521 - 539.

[0011] 5. Zipfel C, Kunze G, Chinchilla D, et al. Perception of the bacterial PAMP EF - Tu by the receptor EFR restricts Agrobacterium - mediated transformation. Cell, 2006, 125(4): 749 - 760.

[0012] 6、Kunze G,Zipfel C,Robatzek S,et al.The N terminus of bacterialelongation factor Tu elicits innate immunity in Arabidopsis plant.The PlantCell,2004,16:3496-3507.

[0013] 7、Lee SW,Han S,Sririyanum M,et al.A type-I secreted,sulfated peptidetriggers XA21-mediated innate immunity.Science,2009,326:850-853.

[0014] 8、Wang L,Albert M,Einig E,et al.The pattern-recognition receptor COREof Solanaceae detects bacterial cold-shock protein.Nature Plants,2016,2:16185.

[0015] 9、Saur IML,Kadota Y,Sklenar J,et al.NbCSPR underlies age-dependentimmune responses to bacterial cold shock protein in Nicotianabenthamiana.Proceedings of the National Academy of Sciences,2016,113:3389–3394.

[0016] 10、Shimizu,T.et al.Two LysM receptor molecules,CEBiP and OsCERK1,cooperatively regulate chitin elicitor signaling in rice.Plant J.2010,64,204–214.

[0017] 11. Hayafune, M. et al. Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-typedimerization. Proc. Natl Acad. Sci. 2014, USA 111, E404–E413.

[0018] 12. Liu, B. et al. Lysin motif-containing proteins LYP4 and LYP6 playdual roles in peptidoglycan and chitin perception in rice innateimmunity. Plant Cell, 2012, 24, 3406–3419.

[0019] 13. Ao, Y. et al. OsCERK1 and OsRLCK176 play important roles inpeptidoglycan and chitin signaling in rice innate immunity. Plant J. 2014, 80, 1072–1084. Summary of the Invention

[0020] This invention reveals that transgenic rice overexpressing the OsLIKE1 gene exhibits significant resistance to rice blast fungus, while conversely, rice with OsLIKE1 gene knockout mutations displays a susceptible phenotype. This leads to the completion of this invention.

[0021] The present invention first provides a receptor kinase OsLIKE1 gene for rice resistance to rice blast fungus, the amino acid sequence of which is shown in SEQ ID NO:3.

[0022] This invention provides a receptor-like kinase OsLIKE1 gene for rice resistance to rice blast fungus, the nucleotide sequence of which is shown in SEQ ID NO:1, or the cDNA nucleotide sequence of which is shown in SEQ ID NO:2.

[0023] The present invention further provides a protein encoded by the OsLIKE1 gene, a receptor-like kinase for rice blast fungus, the amino acid sequence of which is shown in SEQ ID NO:3.

[0024] This invention provides a method for improving the resistance of rice to rice blast fungus, which involves overexpressing the OsLIKE1 gene, a receptor kinase for rice resistance to rice blast fungus, in rice. The OsLIKE1 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO:3.

[0025] Specifically, the overexpression method is as follows: the OsLIKE1 gene, a receptor kinase for rice resistance to rice blast fungus, is cloned and a plant expression vector is constructed. The plant expression vector is then transferred into Agrobacterium, and rice is inoculated. Overexpression lines or their progeny with rice blast resistance are selected.

[0026] Preferably, the cDNA nucleotide sequence of the rice blast fungus-resistant receptor kinase OsLIKE1 gene is shown in SEQ ID NO:2.

[0027] More specifically, the plant expression vector is pXQ, and the Agrobacterium is EHA105.

[0028] The genetically modified rice obtained by the above methods does not affect normal growth and grain production.

[0029] This invention provides the application of the OsLIKE1 gene, a receptor-like kinase for rice blast fungus, or the protein encoded by the OsLIKE1 gene for rice blast fungus, in the cultivation of rice varieties with enhanced resistance to rice blast fungus.

[0030] The present invention also provides the application of the OsLIKE1 gene, a receptor kinase for rice resistance to rice blast fungus, in the identification of rice varieties or plants with enhanced resistance to rice blast fungus, wherein the rice varieties or plants with enhanced resistance to rice blast fungus are obtained by the above method.

[0031] Regarding the protein encoded by the OsLIKE1 gene, a receptor-like kinase for rice blast fungus in this invention, we found that OsLIKE1 can undergo autophosphorylation. Through phosphorylation modification mass spectrometry identification, we found that threonines at positions 522 and 523 of OsLIKE1 were phosphorylated. After further mutating threonines at positions 522 and 523 to alanine, the phosphorylation of OsLIKE1 disappeared.

[0032] This invention is beneficial for the breeding of disease-resistant rice varieties and provides a basis for the later screening of highly resistant rice varieties. For example, this invention can provide rice that overexpresses the receptor kinase OsLIKE1, and through hybridization, obtain transgenic rice materials with higher resistance. Attached Figure Description

[0033] Figure 1This study investigates the disease resistance of rice plants overexpressing and knocking out the OsLIKE1 gene, a rice receptor kinase. In the figures, A represents Western blotting of OsLIKE1 protein overexpressing plants; B represents the construction of the target sequence for the OsLIKE1 gene knockout mutant obtained using CRISPR / Cas9 technology and the sequencing results of the knockout gene; C represents the resistance and susceptibility of OsLIKE1 gene knockout and overexpressing rice to rice blast fungus; D represents the statistical results of lesion size on leaves in the C adapter; and E represents the relative biomass of rice blast fungus on leaves in Figure C.

[0034] Figure 2 Using Mn 2+ - Phos-tag validation of rice receptor kinase OsLIKE1 autophosphorylation and the effect of phosphorylation site mutations on autophosphorylation Western spectroscopy results. Detailed Implementation

[0035] Example 1: Obtaining rice with OsLIKE1 gene overexpression (a type of rice receptor kinase)

[0036] In the initial stage of the study, we identified more than 40 potential interaction targets with LHCB5, a key protein in rice resistance to rice blast fungus, using IP-mass spectrometry. Interaction analysis between these potential targets and LHCB5 revealed an interaction between rice receptor-like kinase OsLIKE1 and LHCB5. The nucleotide sequence of the OsLIKE1 gene, a receptor-like kinase for rice resistance to rice blast fungus, is shown in SEQ ID NO:1, its cDNA nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:3.

[0037] SEQ ID NO:1:

[0038] CGCACTCCCTGCAGGCTCTAGCACAAGCTTAGCTAGCAACGTGAAATCTTTGTAGGC

[0039] AGTTGAAGCTTATATGTGCTGAGCTCCTACCCTCCAATGCCGTCCATCTCCATGTCCCTCT

[0040] CGCCGACGTCGCCGTGCACCTCGCATCGTCGCCGTCACCGTCGCCGACGACCAGACCA

[0041] GCAACAGTGCCAGCGACGGCGGCTGCTGCCCAACTCCAACAACTAAACTGTTCAACTC

[0042] CTTCAGCTACTACTTCACTTGGGTTAATTTGTACATTGCTCTCTGCCATGGCGGCGCCGG

[0043] CAGCCGTTCTTGCCGTGCTCCGGTGGCGGCAGCGGCGGTGGCTGGTTGAGCTGGTCGCT

[0044] CTCCTGCTTCTTCTTGCTCGAGGTGGCGCTGCCGCGGTTGACCAATTCTTGTTTCCCGGC

[0045] TTCTCCGGCGACGGCGTGGCCACCAGCGGCGCGGCCGCCGTCACCTCCACCGGTCTACT

[0046] GCAGCTCACCAACGAGACGAAGGAGGTGTTCGGCCATGGCTTCTACCCCAAGCCGGTC

[0047] AGCTTGAGAAACGCCTCCACCGGCGCGGCGGCCTCCTTCTCCACCACCTTCGTGTTCGC

[0048] CATCGTGCCCAAGTATCCCGACGCGCATGGCCACGGCCTCGCCTTCGCGCTGGCGCCGT

[0049] CGGTGGCCGTCCCCGGCGCCGTCGCCGGCAAGTACCTGGGGCTCTTCAACACGTCCGA

[0050] CAGCACGGGCCAGATCAAGAACAAGATCGTCGCCGTCGAGCTCGACACGGCGCGGGAC

[0051] GACGAGTTCGCCGACATCGACGACAACCACGTCGGCATCGACGACAACAGCCTGAAAT

[0052] CCGTGAACTCCAGCCCGGCGGGGTACCACGACGCCGCCACCGGCGGCAAGCTCGCCAG

[0053] CGTCAACCTGATCGGCGGCGAGCCCCTGCAGGTGTGGATCGAGTACGACGGCGACAGC

[0054] GCGTGGCTCGAGGTGACGGTGTCGCCGGCCGGCATGCTCAGGCCGGCCGCCCCGCTGG

[0055] TGTCTTGCACCGTCAACCTGTCGTCCGCCGTGGCCGCGACACGTACGTCGGGTTCTCG

[0056] GCGGCGAACGGCGCCGCCGGAGCTCGCACTACGTCCTCGGCTGGAGCTTCCGCCTCG

[0057] GCGGCGGCGGCCGCGCGCAGGACCTCGACCTCGCCAAGCTCCCGCGGCTCCCGTCGCC

[0058] GAGCAAACCCAAGAAGACGTTGCCGCCGCTGATCATTCTTGACAATTCTCCTCCTCTCCG

[0059] TCGTGATCCTGCTCCTGGCGGCGGCGGCGTGGCGGCGCTCGTGGTGCGAAGCCGGCG

[0060] GTACGCCGAGAGAGAGGAGGAATGGGAGATCGAGTACGGGGCCACACAGGATCAGCTAC

[0061] AAGGACCTCCACGGCGCGACCAAGGGGTTCCGCGACGTCCATCGGCGCCGCGGCTTCG

[0062] GCAGCGTGTACCACGGCGTGCTGCCGAGGTCCGGCGTGGAGGTCGCCGTGAAGAAGGT

[0063] GTCGCACGACTCGCGGCCAGGGGCTGCGGGAGTTCCGTGTCGGAGATCGCCAGCATGAGC

[0064] CGGCTGCGCCCACCGCAACCTGGTGCAGCTGCTCGGCTACTGCCGGCGCCGCGGCGAGC

[0065] TGGTGCTCGTCTACGACTACATGGCGAACGGCAGCCTCGACAAGCACCTGTTCGCCGGC

[0066] GGCGAGCGGCCGGCGCTGAGCTGGGAGAAGCGCGGCAAGATCGTCCGGGACGTCGCC

[0067] GCCGGGCTGCTGTACCTGCACGAGGGGTGGGAGCAGGTGGTGGTGCACCGTGACATCA

[0068] AGGCCAGCAACGTGCTCCTCGACGCCGACATGAACGGCAAGCTCAGCGACTTCGGCCT

[0069] CGCCCGGCTCTACGACCACGGCGCTAACCCGCAGACGACGCGCATCGTCGGCACGCTC

[0070] GGCTACCTCGCGCCGGAGCTGAGCAAGACCGGCAAGGCCACCACGAGCACCGACGTCT

[0071] TCGCCTTCGGCGCCTTCTTGCTCGAGGTGGCGTGCGGCCGGAGGCCCATGGAGTTCACC

[0072] GTCGACGACGACTCGCCGGGGCTCGTCGAGCTCGTCCTCGAGCACTGGAAGGCCGGCG

[0073] AGATCACGGCGGCGAGGGACCCCAGGATCGGCGACTGTGACGAGGACGATCTCGAGGT

[0074] CGTCCTCAAGCTCGGGCTTCTCTGCTCGCACCCTGACCCGCGCCGCCGGCCGAGCATGA

[0075] GGCAGGTGGTGCAGATCTTGGAAGGCGCCGCGCCGGCGCCGGAGACGTTGCCGGAGG

[0076] ACCTAGAATGCGGCGTGGGGCAGTTCTACGACGAGTCATTCGACGAGTTCGTCACCGGC

[0077] TTCCCGTCCACGTCGGAGATCACCACGTCGACGACGCAGTCCACCGACGAGCAGCAGC

[0078] GGCTGGTTGGCTGTGTTCAGTTGAGCACTGCCGATTTTCTGAAGACAACTTAAAATGTA

[0079] AAAATGTCAGTGAGCAAATACTTGCAGCATTAGGTGATTGTTTGGGCTTTGGATCTTCTG

[0080] GAGTTAGTAACATAGGGATACTGTCAATCATTTTCAGATATGATCATATAGAGGTTTTGAT

[0081] CAAAGATTAGTACAGCATTCAGCTTAGAAGTTACCTTGTTGCAGTTTCAGAAGAATGTAC

[0082] ACCTGCAAGAAGGCAAGAACCGTGTAAGAAATTTTGCAAGGGAAGAAAAAAAAATATG

[0083] GATCCCCTTTTTTTTTTTGTAGCTGCATCTTTCTTGGGGCAGTTTGCTGTTGAGCCAGATC

[0084] AGAATATTAGCCAATAGACTGATCACTGATGTCGATTCTGTCATGCAGAAAACATGCGAT

[0085] ATATAATATGACATATTGGCACCTTGCTCAAAAGGAACCAAAGATATTGCAGCCTGCTTC

[0086] TCATCATGGGAAAAAGACAATGAAAACCTAAAAAAATGCTGTCCTAATGTTCTGCTTTTA

[0087] CAACAAAAGATGAACATGACAAATATTAGCAAGGTATCAAACAAAAAGATGACAACAA

[0088] ATGGAGACAGTTCAGGCCATGGTTCTAGGGATTCTTCTGATTCATACGAGAACTGAACG

[0089] GGCTTTCTTGTGTTCCAAACAGAGGACATTAGATTGGTTGCCGAGGAAAAAAGAACTAC

[0090] TTGCAAAATGTTGACAACTGACAAGCAATGCCAGATTGTGTCCATGGCTCCATGCAACA

[0091] TTAGTTTACATCCTATCCAAGCTAAAACTAGCATCCCTACTACCCAATCGTAATGACTCCA

[0092] CCTATAAGCACAGGTGACGGCTCTCTTCCTCAGAGTTATTCATCATCCTCCTGAGTTCCCCGAGAAGCACCTTGGATCATTTTGTCATCAACTCCTGCGATTAAG。

[0093] SEQ ID NO:2:

[0094] ATGGCGGCGCCGGCAGCCGTTCTTGCCGTGCTCCGGTGGCGGCAGCGGCGGTGGCT

[0095] GGTTGAGCTGGTCGCTCTCCTGCTTCTTCTTGCTCGAGGTGGCGCTGCCGCGGTTGACC

[0096] AATTCTTGTTTCCCGGCTTCTCCGGCGACGGCGTGGCCACCAGCGGCGCGGCCGCCGTC

[0097] ACCTCCACCGGTCTACTGCAGCTCACCAACGAGACGAAGGAGGTGTTCGGCCATGGCTT

[0098] CTACCCCAAGCCGGTCAGCTTGAGAAACGCCTCCACCGGCGCGGCGGCCTCCTTCTCCA

[0099] CCACCTTCGTGTTCGCCATCGTGCCCAAGTATCCCGACGCGCATGGCCACGGCCTCGCCT

[0100] TCGCGCTGGCGCCGTCGGTGGCCGTCCCCGGCGCCGTCGCCGGCAAGTACCTGGGGCT

[0101] CTTCAACACGTCCGACAGCACGGGCCAGATCAAGAACAAGATCGTCGCCGTCGAGCTC

[0102] GACACGGCGCGGGACGACGAGTTCGCCGACATCGACGACAACCACGTCGGCATCGACG

[0103] ACAACAGCCTGAAATCCGTGAACTCCAGCCCGGCGGGGTACCACGACGCCGCCACCGG

[0104] CGGCAAGCTCGCCAGCGTCAACCTGATCGGCGGCGAGCCCCTGCAGGTGTGGATCGAG

[0105] TACGACGGCGACAGCGCGTGGCTCGAGGTGACGGTGTCGCCGGCCGGCATGCTCAGGC

[0106] CGGCCGCCCCGCTGGTGTCTTGCACCGTCAACCTGTCGTCCGCCGTGGCCGGCGACACG

[0107] TACGTCGGGTTCTCGGCGGCGAACGGCGCCGCCGCGAGCTCGCACTACGTCCTCGGCTG

[0108] GAGCTTCCGCCTCGGCGGCGGCGGCCGCGCGCAGGACCTCGACCTCGCCAAGCTCCCG

[0109] CGGCTCCCGTCGCCGAGCAAACCCAAGAAGACGTTGCCGCCGCTGATCATCTTGACAAT

[0110] TCTCCTCCTCTCCGTCGTGATCCTGCTCCTGGCGGCGGCGGCCGTGGCGGCGCTCGTGG

[0111] TGCGAAGCCGGCGGTACGCCGAGGAAGAGGAGGAATGGGAGATCGAGTACGGGCCAC

[0112] ACAGGATCAGCTACAAGGACCTCCACGGCGCGACCAAGGGGTTCCGCGACGTCATCGG

[0113] CGCCGGCGGCTTCGGCAGCGTGTACCACGGCGTGCTGCCGAGGTCCGGCGTGGAGGTC

[0114] GCCGTGAAGAAGGTGTCGCACGACTCGCGGCAGGGGCTGCGGGAGTTCGTGTCGGAGA

[0115] TCGCCAGCATGAGCCGGCTGCGCCACCGCAACCTGGTGCAGCTGCTCGGCTACTGCCGG

[0116] CGCCGCGGCGAGCTGGTGCTCGTCTACGACTACATGGCGAACGGCAGCCTCGACAAGC

[0117] ACCTGTTCGCCGGCGGCGAGCGGCCGGCGCTGAGCTGGGAGAAGCGCGGCAAGATCGT

[0118] CCGGGACGTCGCCGCCGGGCTGCTGTACCTGCACGAGGGGTGGGAGCAGGTGGTGGTG

[0119] CACCGTGACATCAAGGCCAGCAACGTGCTCCTCGACGCCGACATGAACGGCAAGCTCA

[0120] GCGACTTCGGCCTCGCCCGGCTCTACGACCACGGCGCTAACCCGCAGACGACGCGCATC

[0121] GTCGGCACGCTCGGCTACCTCGCGCCGGAGCTGAGCAAGACCGGCAAGGCCACCACGA

[0122] GCACCGACGTCTTCGCCTTCGGCGCCTTCTTGCTCGAGGTGGCGTGCGGCCGGAGGCCC

[0123] ATGGAGTTCACCGTCGACGACGACTCGCCGGGGCTCGTCGAGCTCGTCCTCGAGCACT

[0124] GGAAGGCCGGCGAGATCACGGCGGCGAGGGACCCCAGGATCGGCGACTGTGACGAGG

[0125] ACGATCTCGAGGTCGTCCTCAAGCTCGGGCTTCTCTGCTCGCACCCTGACCCGCGCCGC

[0126] CGGCCGAGCATGAGGCAGGTGGTGCAGATCTTGGAAGGCGCCGCGCCGGCGCCGGAGA

[0127] CGTTGCCGGAGGACCTAGAATGCGGCGTGGGGCAGTTCTACGACGAGTCATTCGACGA

[0128] GTTCGTCACCGGCTTCCCGTCCACGTCGGAGATCACCACGTCGACGACGCAGTCCACCG

[0129] ACGAGCAGCAGCGGCTGGTTGGCTGTGTTCAGTTGAGCACTGCCGATTTTCTGAAGACAACTTAA。

[0130] SEQ ID NO:3:

[0131] MAAPAAVLAVLRWRQRRWLVELVALLLLLARGGAAAVDQFLFPGFSGDGVATSGAAA

[0132] VTSTGLLQLTNETKEVFGHGFYPKPVSLRNASTGAAASFSTTFVFAIVPKYPDAHGHGLAFA

[0133] LAPSVAVPGAVAGKYLGLFNTSDSTGQIKNKIVAVELDTARDDEFADIDDNHVGIDDNSLKS

[0134] VNSSPAGYHDAATGGKLASVNLIGGEPLQVWIEYDGDSAWLEVTVSPAGMLRPAAPLVSCT

[0135] VNLSSAVAGDTYVGFSAANGAAASSHYVLGWSFRLGGGGRAQDLDLAKLPRLPSPSKPKK

[0136] TLPPLIILTILLLSVVILLLAAAAVAALVVRSRRYAEEEEEWEIEYGPHRISYKDLHGATKGFR

[0137] DVIGAGGFGSVYHGVLPRSGVEVAVKKVSHDSRQGLREFVSEIASMSRLRHRNLVQLLGYC

[0138] RRRGELVLVYDYMANGSLDKHLFAGGERPALSWEKRGKIVRDVAAGLLYLHEGWEQVVV

[0139] HRDIKASNVLLDADMNGKLSDFGLARLYDHGANPQTTRIVGTLGYLAPELSKTGKATTSTD

[0140] VFAFGAFLLEVACGRRPMEFTVDDDSPGLVELVLEHWKAGEITAARDPRIGDCDEDDLEVV

[0141] LKLGLLCSHPDPRRRPSMRQVVQILEGAAPAPETLPEDLECGVGQFYDESFDEFVTGFPSTSEITTSTTQSTDEQQRLVGCVQLSTADFLKTT.

[0142] This invention utilizes homologous recombination to construct the coding region of the target gene OsLIKE1 into a pXQ vector. During primer design, homologous arms near the SmaI restriction site in the pXQ vector are first added to the upstream and downstream primers for OsLIKE1 to amplify the OsLIKE1 gene. Then, the target gene and the linearized pXQ vector digested with SmaI are ligated using homologous recombination enzyme to form the pXQ-OsLIKE1 vector. Because the vector contains a built-in flag tag, it can be used for later validation. The pXQ-OsLIKE1 vector is transformed into Agrobacterium EHA105 via chemical transformation, and positive single colonies are selected to prepare OD. 600Rice callus was infected with a 0.2% inoculum solution for 15 minutes, then cultured at 20°C for 48-72 hours. Single-clone callus tissue was then picked and transferred to a selection medium, cultured in the dark at 26°C. After 25 days, positive single-clone callus tissue was selected and subcultured in the dark at 26°C. After 7-10 days of culture, the positive callus tissue was inoculated onto a differentiation medium and cultured at 27°C under light for 15 days. Then, 3-5cm differentiated shoots were inoculated onto a rooting medium and cultured at 30°C under light for 7 days. When the seedlings reached about 8cm in length, plants with well-developed root systems and normal growth were selected and cultured indoors for 7 days before being transferred to the field for propagation. After two propagation cycles, plants overexpressing the OsLIKE1 gene were obtained. Two overexpression lines were selected from the obtained transgenic plants, and Western spectroscopy was used to detect the overexpression of this gene in rice. Figure 1 (A) This demonstrates that the obtained transgenic plants can be used for subsequent experiments.

[0143] Example 2: Obtaining rice with OsLIKE1 gene knockout, a rice receptor kinase.

[0144] This invention utilizes CRISPR / Cas9 technology to first construct an OsLIKE1 knockout vector, then transforms the knockout vector into Agrobacterium EHA105 via chemical transformation, and selects positive single colonies to prepare OD. 600 Rice callus was infected with a 0.2% inoculum solution for 15 minutes, then cultured at 20°C for 48-72 hours. Single-clone callus tissue was then picked and transferred to a selection medium, cultured in the dark at 26°C. After 25 days, positive single-clone callus tissue was selected and subcultured in the dark at 26°C. After 7-10 days of culture, the positive callus tissue was inoculated onto a differentiation medium and cultured at 27°C under light for 15 days. Then, 3-5cm differentiated shoots were inoculated onto a rooting medium and cultured at 30°C under light for 7 days. When the seedlings reached about 8cm in length, plants with well-developed root systems and normal growth were selected and cultured indoors for 7 days before being transferred to the field for propagation. After two propagation cycles, a knockout mutant of the OsLIKE1 gene was obtained. Sequencing confirmed that the gene had been knocked out, and two different mutations were present. Figure 1 (B)

[0145] Example 3: Analysis of resistance and susceptibility between rice varieties overexpressing and knockout of the rice receptor kinase OsLIKE1 gene.

[0146] This invention involves propagating the obtained overexpression and knockout transgenic rice plants to obtain third-generation stably inherited progeny plants for pathogenicity testing. The spores of the wild-type strain Guy11 of *Pseudomonas aeruginosa* were inoculated as a foliar spray on rice plants; the concentration of Guy11 spores was 5 x 10⁻⁶.4 The OsLIKE1 gene knockout mutant was found to be susceptible to the disease, while the OsLIKE1 gene overexpression plants were resistant to Guy11. Figure 1 (C, D); further, by measuring the relative biomass of rice blast fungus on rice leaves, the contribution of the OsLIKE1 gene to the rice's resistance to rice blast fungus was further demonstrated. Figure 1 (E).

[0147] Example 4: Identification and Verification of Phosphorylation Sites of Rice Receptor Kinase OsLIKE1

[0148] This invention utilizes homologous recombination to construct the coding region of the target gene OsLIKE1 into the pET32a(+) vector. During primer design, homologous arms near the EcoRI or HindIII restriction sites in the pET32a(+) vector are first added to the upstream and downstream primers of OsLIKE1 to amplify the OsLIKE1 gene. Then, the target gene and the linearized pET32a(+) vector, which has been double-digested with EcoRI and HindIII, are ligated using a homologous recombination enzyme to construct the pET32a(+)-OsLIKE1 vector. Since the vector contains a His tag, it can be used for later validation. Subsequently, the constructed vector is transformed into the prokaryotic expression strain BL21(DE3), and the protein is induced under conditions of 0.3 Mm IPTG, 16℃, and 220 rpm. The OsLIKE1 protein is purified by using the His tag on the OsLIKE1 protein. An appropriate amount of purified OsLIKE1 protein was added to a kinase reaction buffer containing: 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM CaCl2, 10 μM ATP, and 1 mM DTT. After preparation, the reaction mixture was incubated at 30°C for 45 minutes. Immediately afterwards, an appropriate amount of protein loading buffer was added to the reacted sample, and the mixture was denatured at 95°C for 5 minutes to complete the protein loading sample preparation. Then, Mn... 2+ The protein samples were separated using a phos-tag gel, and then Western spectroscopy was used to analyze the migration of OsLIKE1 protein on a PVDF membrane to determine whether phosphorylation had occurred. The results showed that OsLIKE1 was phosphorylated. Figure 2 (From left to right, lanes 1 and 2). Then, we performed phosphorylation modification mass spectrometry analysis on the phosphorylated samples and found that threonine residues at positions 522 and 523 of the receptor-like kinase OsLIKE1 were phosphorylated. Further mutation of threonine residues at positions 522 and 523 to alanine revealed that phosphorylation of the threonine mutant proteins at positions 522 and 523 of the receptor-like kinase OsLIKE1 still existed. Figure 2 (From left to right, lanes 3 and 4), when the threonines at positions 522 and 523 are simultaneously mutated to alanine, the phosphorylation of OsLIKE1 disappears. Figure 2 The image (lane 5 from left to right) shows that threonine residues at positions 522 and 523 of OsLIKE1 are key phosphorylation sites. Activation of this phosphorylation site can induce phosphorylation of LHCB5, a key protein in rice resistance to rice blast fungus, thereby inducing rice resistance to rice blast fungus (CN201810028068.9). The identification of the phosphorylation site of the receptor-like kinase OsLIKE1 provides a theoretical basis for the molecular selection of rice materials resistant to rice blast fungus. Therefore, we can utilize the phosphorylation site of the receptor-like kinase OsLIKE1 to provide high-quality gene resources for rice disease resistance breeding, and simultaneously use molecular cloning techniques to screen and cultivate disease-resistant and high-yielding rice plants.

Claims

1. A method for improving the resistance of rice to rice blast fungus, characterized in that, The OsLIKE1 gene, a receptor-like kinase for rice resistance to rice blast fungus, was overexpressed in rice. The OsLIKE1 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO:

3.

2. The method according to claim 1, characterized in that, The overexpression method is as follows: the OsLIKE1 gene, a receptor kinase for rice resistance to rice blast fungus, is cloned and a plant expression vector is constructed. The plant expression vector is then transformed into Agrobacterium, and rice is inoculated. Overexpression lines or their progeny with rice blast resistance are selected.

3. The method according to claim 1 or 2, characterized in that, The cDNA nucleotide sequence of the OsLIKE1 gene, a receptor-like kinase for rice blast fungus, is shown in SEQ ID NO:

2.

4. The method according to claim 3, characterized in that, The plant expression vector is pXQ.

5. The method according to claim 4, characterized in that, The Agrobacterium is EHA105.

6. Application of the OsLIKE1 gene, a receptor-like kinase for rice resistance to rice blast fungus, or its encoded protein, in the breeding of rice varieties with enhanced resistance to rice blast fungus; The amino acid sequence encoded by the OsLIKE1 gene, a receptor-like kinase for rice blast fungus, is shown in SEQ ID NO:

3.

7. The application of the OsLIKE1 gene, a receptor-like kinase for rice blast fungus, in identifying rice varieties or plants with enhanced resistance to rice blast fungus, wherein the rice varieties or plants with enhanced resistance to rice blast fungus are obtained by the method described in any one of claims 1 to 5.

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